The present invention relates generally to the treatment of fibrotic conditions, and to the use of antifibrotic agents for the amelioration and modification of such diseases. The present invention is also concerned with therapeutic compositions in which antifibrotic agents are chemically combined with carriers such as polymers in order to enhance the pharmacokinetic profile of the antifibrotic agents.
The fibrotic conditions which the present invention is intended to treat include changes in the structure and function of various organs in connection with the metabolism of collagen and other biomolecules. One of the long-term sequelae of hypertension is the deposition of connective tissue in walls of blood vessels. In hypertensive rats, collagen biosynthesis and deposition are increased in the aorta, and these effects are reversed when blood pressure is lowered by antihypertensive drugs. Treatment of animals with experimental hypertension with agents that selectively inhibit collagen formation and reduce vascular collagen content, suggesting that increased collagen contributes to the maintenance of hypertension. Although the use of antifibrotic agents has increased the understanding of the role of collagen in hypertension and vascular disease, their application as potential therapeutic agents for chronic conditions has been limited.
Collagen is the most abundant protein in vertebrates. The biosynthesis of collagen involves unique post-translational modification of pro-alpha chains. Hydroxylation of prolyl and lysyl residues, a key part of collagen formation, is vital for normal triple-helix formation and intermolecular cross-linking. When post-translational processing is inhibited, non-helical procollagen forms, and it is then degraded by intracellular proteases and is secreted into the extracellular matrix at a slower rate as a nonfunctional protein. The incorporation of proline analogues, e.g., cis-4-hydroxy-L-proline (cHyp), into nascent pro-alpha chains reduces the extracellular accumulation of collagen. The agents described herein are believed to act more generally by inhibiting collagen synthesis and thereby averting certain of the pathophysiological sequelae of fibrosis, such as atherosclerosis and hypertension. Through the distortion of bond angles and from steric hindrance among polypeptide chains; cHyp inhibits the folding of pro-alpha chains into a stable triple helix. Other proline analogues such as cis-4-fluoroproline, cis-4-bromoproline, and 3,4-dehydroproline have similar effects, but can also inhibit other post-translational steps. The compound 3,4-dehydroproline is an example of a proline analogue which can also inhibit other post-translational steps; for example, 3,4-dehydroproline inhibits prolyl hydroxylase activity. This proline analogue has been administered to humans with pulmonary fibrosis in the condition referred to as adult respiratory distress.
The antifibrotic agents described herein are most effective in tissues undergoing rapid rates of collagen synthesis. For example, collagen comprises about one-third of the dry weight of pulmonary arteries in which synthesis increases rapidly following induction of hypertension. Exposure to hypoxia causes constriction of small pulmonary arteries and hypertension develops form sustained vasoconstriction and structural changes in the vascular wall. Proliferation of vascular smooth muscle cells and connective tissue accumulation thicken the vessel walls and narrow the lumen of pulmonary arteries. These structural changes cause or contribute to hypertension.
Collagen metabolism has been implicated as a negative factor in other diseases and conditions. For example, scar tissue is comprised largely of collagen. While some scar tissue is normal as a result of the closure and healing of wounds, excess scar tissue and collagen based adhesions are often undesirable and unhealthy. It is important to note, accordingly, that several proline analogues have been shown to be effective in inhibiting scar formation.
The present invention in particular relates to polymers which contain the antifibrotic compounds described herein, pharmaceutical compositions containing such polymers and various methods of preparation and use. In such polymers, cis-hydroxyproline (cHyp) or another antifibrotic agent is the pharmacologically active agent, useful in controlling the proliferation of collagen or the other changes in tissue as described herein in detail. This is particularly important in diseases and conditions where collagen is deposited or synthesized in abnormally high levels, or where collagen is not properly broken down or removed, contributing to the pathology of the particular disease or condition. In the past it has been recognized that cHyp is active in reducing the abnormal proliferation of collagen. More particularly, the pharmacological effectiveness of cHyp has been demonstrated in treating pulmonary fibrosis. Unfortunately, it is also recognized that cHyp can be potentially toxic if used improperly, particularly in chronic use, and thus has had limited clinical utility.
In recent efforts to provide a stable carrier for cHyp, poly tethylene glycol-co-lysine) (PEG-Lys) functioned as such a carrier for the antifibrotic agent; Poiani et al., Bioconjugate Chemistry; 1994; 5(6):621-630. It was demonstrated that a hydrolytically stable amide-linkage between cHyp and the polymeric backbone is needed to maximize the antifibrotic activity both in vitro and in vivo; Poiani, G. J., et al., supra. Typically, the cHyp is coupled to the free acid carrier via the dicyclohexylcarbodiimide, 4-dimethylaminopyridine (DCC/DMAP) system. However, the primary disadvantage of this system is the significant variability in cHyp attachment. The maximum degree of attachment via this coupling scheme for the amide-linked cHyp is approximately 65%, requiring a three-fold excess of the appropriately protected cHyp. In order to alleviate this variability and low degree of drug incorporation, the present invention uses the dipeptide of L-Lys and cHyp as the drug-containing chain extender. Thus, controlled dosage forms, i.e., mg/ml of a carrier matrix for which a specific drug content is maintained, can be readily obtained and administered.
The present invention thus provides an improved synthetic scheme that has been developed in order to optimize the capacity of cHyp that can be conjugated to the poly(PEG-Lys) carrier, and a detailed hydrolytic stability profile has been developed. In a further extension of the present invention, aimed at combining the high bioactivity of poly(PEG-Lys-cHyp) which has been observed with further extensions of existing treatments into fibrotic lung disorders, there is also provided intravenous liposomal delivery of drug conjugates using non-immunogenic polysaccharide-coated vesicles. Organ distribution and biological stability were investigated using radiolabeled drug conjugates of the present invention.
The controlled release and targeting of drugs to specific cells and organs has become increasingly important. Accordingly, the present invention provides a hybrid drug delivery system comprising a non-specific, non-cytotoxic, polymeric carrier containing a covalently bound, low molecular weight, water soluble, polar drug delivered by means of a liposomal vehicle containing target-specific ligands. Data has been gathered and is presented below in order to demonstrate the efficacy of this drug delivery system, as well as to illuminate the general principles on which it operates. The targeting of such sustained release antifibrotic treatment compositions to tissues with increased collagen production is an approach which can be taken in order to prevent organ fibrosis. Broader applications are found in treating scar for mnation, adhesions, and fibrosing disorders of other visceral organs.
Accordingly, the present invention seeks to overcome the disadvantages of past approaches to treatment of fibrotic diseases. Thus, one object of the present invention is to facilitate the use of antifibrotic agents in the treatment of diseases and conditions in which collagen metabolism is to be modified, such as when excess collagen synthesis or deposition occurs.
Another object of the present invention is to combine the antifibrotic agents described herein with other compounds, e.g., polymers, to improve the pharmacokinetic profile of these drugs.
Another object of the present invention is to combine the therapeutic agents with compounds which have little if any toxicity or side effects of their own.
Another object of the present invention is to enhance the delivery of the antifibrotic agents to the site of activity.
Another object of the present invention is to provide antifibrotic agents in a variety of polymeric and monomeric forms which can be used to modify the pharmacokinetic profile of the agent in question.
These and other objects will be apparent to those of ordinary skill in the art from the teachings which follow.
The publications enumerated further below are illustrative of the state of the art which encompasses the above-defined field of the invention. Each said publication is hereby incorporated herein by reference, each in its entirety:
Abuchowski et al., J. Biol. Chem., 1977, 252(11):3578;
Ajisaka et al., Biochem. Biophys. Res. Commun., 1980, 97(3):1076;
Bowers-Nemia et al., Heterocycles, 1983, 20(5):817;
Zalipsly et al., Eur. Polym. J., 1983, 19 (12):1177;
Kohn et al., J. Am. Chem. Soc., 1987, 109:817;
Ouchi et al., J. Macromol. Sci.xe2x80x94Chem., 1987, A24(9):1011;
Yamsuki et al., Agric. Biol. Chem., 1988, 52:2185-2196;
Nathan et al., J. Polym. Preprints 1990, 1990, 31(2):213;
Papaioannu et al., Acta Chem. Scand., 1990, 44:243;
Poiani et al., Amino Acids: Chem. Biol. and Med., Lubec and Rosenthal, eds., 1990, 634-642;
Poiani et al., J. Appl. Physiol., 1990, 68:1542;
Somak et al., Free Rad. Res. Commun., 1991, 12-13:553-562;
Zalipsky et al., xe2x80x9cIn Polymeric Drugs and Drug Delivery Systemsxe2x80x9d, Dunn and Ottenbrite, eds., Am. Chem. Soc., 1991, 469:91;
Ertel et al. In Polym. Mat. Sci. Eng. American Chem. Soc., 1992, 66:486;
Nathan et al., Macromolecules, 1992, 25:4476-4484;
Roseng, et al., J. Biol. Chem., 1992, 267(32):22981-22993;
Nathan et al., Bioconjugate Chem., 1993, 4:54-
Nathan et al., J. Bioact. Compat. Polym., 1994, (in press);
Poiani et al., Bioconjugate Chem., 1994, 5(6):621-630;
Monfardini et al., Bioconjugate Chem., 1995, 6:62-69;
Zalipsky, Bioconjugate Chem., 1995, 6(2):150-165;
In accordance with the present invention, an antifibrotic composition is disclosed which comprises one or more dipeptides consisting of an L-proline or derivative antifibrotic agent comprising one or more members selected from the group consisting essentially of 3,4-dehydro-L-proline and laevo and cis isomers of compounds of the general structural formula: 
wherein R is OH, Cl, F, NH2, SH, SCH3, OCH3, ONO2, OSO2, OSO3H, H2PO4, or COOH; and pharmaceutically acceptable salts thereof; said L-proline or derivative antifibrotic agent being covalently bound to L-lysine to form each dipeptide, which in turn is covalently bound to a polymer comprising one or more monomers or prepolymers selected from the group consisting essentially of ethylene glycol, propylene glycol, butylene glycol, isobutylene glycol, and povidone to form a copolymer conjugate; wherein said antifibrotic composition is prepared by covalently binding said L-proline or derivative antifibrotic agent to said L-lysine to form one or more said dipeptides, and thereafter covalently binding said dipeptide to said polymer to form said copolymer conjugate, wherein said formation of said copolymer conjugate proceeds to give in excess of a 98% yield.
In particular, the present invention provides an antifibrotic composition wherein the L-proline or derivative antifibrotic agent is cis-4-hydroxyproline, and the polymer is poly(ethylene glycol) having a weight average molecular weight of from about 500 to about 15,000.
The present invention also provides intermediates useful in the process of making the copolymer conjugates. These intermediates comprise the dipeptides consisting of an L-proline or derivative antifibrotic agent as defined above, covalently bound to L-lysine to form each dipeptide. Said intermediates have the following formula: 
wherein R1 is a conventional amine protecting group; and R is OH, Cl, F, NH2, SH, SCH3,OCH3,ONO2, OSO2, OSO3H, H2PO4, or COOH; and pharmaceutically acceptable salts thereof.
There is further provided a method of preparing the antifibrotic composition described above, comprising covalently binding said L-proline or derivative antifibrotic agent to said L-lysine to form one or more said dipeptides, and thereafter covalently binding said dipeptide to said polymer to form a copolymer conjugate, under conditions which do not substantially reduce the pharmacological activity of the antifibrotic agent, and wherein said formation of said polymer conjugate proceeds to give in excess of a 98% yield. In particular, the Nxcex1- and Nxcex5-termini of the L-lysine are protected, e.g., with t-butoxycarbonyl, or other suitable amine protecting groups; and the N-hydroxysuccinimide ester of the L-lysine is used in the coupling reaction, along with conventional coupling agents, e.g., dicyclohexylcarbodiimide (DCC) together with dimethylaminopyridine (DMAP). After formation of the dipeptide, one or more thereof are then covalently bound to said polymer comprising one or more monomers or prepolymers selected from the group consisting essentially of ethylene glycol, propylene glycol, butylene glycol, isobutylene glycol, and povidone to form said copolymer conjugate. In this coupling reaction, the terminal hydroxyl groups of, e.g., poly(ethylene glycol), are activated with conventional activating groups, e.g., succinimide to form the bis(succinimidyl)carbonate of the polymer. Amide linkages are then formed between the dipeptide units and the polymer units by using conventional polymerization promoters, e.g., sodium bicarbonate.
The copolymer conjugates described above can be included in a pharmaceutical composition in combination with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any of those commonly recognized vehicles used in the formulation of pharmaceutical products.
Another aspect of the invention involves a pharmaceutical composition as described above, wherein the copolymer conjugate is used in, and as a part of, the pharmaceutically acceptable carrier, and thus serves as a carrier molecule for delivery of the antifibrotic active agent, while at the same time serving as a component of the delivery vehicle. Furthermore, the vehicle itself has a site specific makeup recognized by receptors in various organ tissues where the antifibrotic agents will be effective. A preferred embodiment of this dual use is a liposomal vehicle, e.g., PEG-conjugated liposomes, and additionally, liposomes coated with cholesterol derivatized amylopectin, wherein the antifibrotic copolymer conjugate is entrapped within said liposomes.
The invention also encompasses a method of treatment of diseases or conditions wherein abnormal collagen accumulation or proliferation is of concern, comprising administering to a mammalian patient in need of such treatment at least one of the antifibrotic agents described herein as a copolymer conjugate in an amount effective for treating the abnormality in collagen accumulation.
The diseases and conditions in which the antifibrotic agents described herein are particularly useful include pulmonary conditions, such as pulmonary fibrosis; atherosclerotic conditions, such as arteriosclerosis; renal disorders, such as renal hypertension; hepatic disorders, such as cirrhosis; scar formation, adhesions, and fibrosing disorders of other visceral organs; and other like conditions.